Liquid formulation of long-acting human growth hormone immunoglobulin conjugate

ABSTRACT

The present invention relates to a sustained type human growth hormone conjugate preparation comprising: a sustained type human growth hormone (hGH) conjugate resulting from conjugation between the immunoglobulin Fc region and a human growth hormone (hGH) constituting a bioactive peptide; a buffer solution; a nonionic surfactant; and a sugar alcohol. More specifically, the present invention relates to a sustained type human growth hormone conjugate freeze dried preparation and liquid preparation, to a production method for the freeze dried preparation, to a method of reconstituting the freeze dried preparation, and to a kit comprising the freeze dried preparation and a reconstituting solution.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/KR2014/009059 filed Sep. 26, 2014, claiming priority based on KoreanPatent Application No. 10-2013-0115177 filed Sep. 27, 2013.

TECHNICAL FIELD

The present invention relates to a formulation of a long-acting humangrowth hormone conjugate, comprising a long-acting human growth hormone(hGH) conjugate in which the human growth hormone as a physiologicallyactive peptide is linked to an immunoglobulin Fc region, a buffer, anon-ionic surfactant and a sugar alcohol, specifically a lyophilizedformulation and a liquid formulation of a long-acting human growthhormone conjugate, a method for preparing the lyophilized formulation, amethod for reconstituting the lyophilized formulation and a kitcomprising the lyophilized formulation and a solution forreconstitution.

BACKGROUND ART

Human growth hormone (hereinafter referred to as “hGH”) is a polypeptidehormone consisting of 191 amino acids having a molecular weight of about22,000, being secreted from the anterior pituitary gland. The humangrowth hormone has been mostly used for the treatment of pediatricpituitary dwarfism. Conventionally, hGH extracted from the humanpituitary gland has been used but only a very limited number of peoplehave been treated due to its limited supply. Also, since the reports ofCreutzfeldt-Jacob disease, a degenerative neurological disorder, foundin some of the patients treated with the hGH extracted from thepituitary gland, the use of hGH extracted from the pituitary glands hasbeen banned. Currently, the development of genetic engineeringtechniques has enabled production of hGH in E. coli and yeast, and thebiosynthetic hGH medicines produced therefrom have been approved inseveral countries since 1985 and become commercially available afterpassing toxicological and clinical tests.

In general, polypeptides such as hGH have low stability and thus areeasily denatured. Also, they are readily degraded by serum proteases andremoved by the kidneys or liver. Thus, protein drugs containingpolypeptides as pharmaceutical ingredient have to be frequentlyadministered to patients to maintain its blood concentration and titer.However, since the protein drugs are often administered in the form ofinjection, frequent injection of the protein drugs to maintain theoptimal blood concentration of the active polypeptides causes a lot ofpain to the patients. To solve these problems, there have been manyattempts to increase the stability of a protein drug in blood andmaintain its blood concentration at high level for a long period of timeto maximize the therapeutic effects of the medicine.

Recently, Korean Patent No. 10-0567902 (Physiologically ActivePolypeptide Conjugate Having Improved In Vivo Durability) and KoreanPatent No. 10-0725315 (Protein Complex Using An Immunoglobulin FragmentAnd Method For The Preparation Thereof) disclosed conjugates prepared bylinking physiologically active polypeptides with an immunoglobulin Fcregion and a non-peptidyl polymer, as long-acting formulations ofprotein drugs, enabling both a minimal reduction of protein activity andan increase in protein stability. According to these methods, hGH may beused as a physiologically active polypeptide to prepare a long-actinghGH conjugate. For commercializing the drug containing the long-actinghGH conjugate, it is essential to prevent physicochemical changes suchas denaturation, aggregation, adsorption, or hydrolysis due todegradation induced by light, heat or impurities in additives duringstorage and transport processes, while retaining the in-vivo activitiesof hGH. Since the long-acting hGH conjugate has a larger size andincreased molecular weight compared to a hGH polypeptide, it isdifficult to stabilize the conjugate.

Lyophilization (freeze-drying) is commonly used to preserve proteins byremoving water from the protein preparation of interest. Lyophilizationis a process by which the material to be dried is first frozen and thenthe ice or frozen solvent is removed by sublimation. An excipient may beincluded in a pre-lyophilized formulation to maintain or enhance proteinstability during the lyophilization process or to improve stability ofthe lyophilized product during storage. However, the composition of alyophilized formulation applicable to one protein is often notapplicable to other proteins due to the difference in properties of theproteins to be preserved. Specifically, different proteins may beinactivated under different conditions during the storage,lyophilization and reconstitution processes owing to their differentchemical properties. That is, the enhancement in stability provided bythe materials used for stabilization is not identical for differentproteins and, accordingly, the suitable ratios, concentrations and kindsof the stabilizers used to provide stability during the storage,lyophilization and reconstitution processes vary depending on thephysicochemical properties of the proteins. When different stabilizersare used in combination, an unwanted negative effect may be derived dueto their competition or adverse reactions and an unexpected effect mayoccur due to the change in the nature or concentration of the proteinduring the lyophilization or storage processes. Therefore, proteinstabilization requires a lot of effort and precautions.

Particularly, since a long-acting hGH conjugate having improved in vivodurability and stability has a form in which the human growth hormone asa physiologically active peptide is linked to the immunoglobulin Fcregion, its molecular weight and volume differs greatly from those ofthe human growth hormone. Therefore, a special composition is requiredfor stabilizing the protein. Also, since each of the physiologicallyactive peptide hGH and the immunoglobulin Fc region has differentphysicochemical properties, they should be stabilized simultaneously.However, as described above, different peptides or proteins may begradually inactivated under different ratios and conditions due to thedifference in their physicochemical properties. Also, when stabilizerssuitable for each peptide or protein are used simultaneously, they maycause adverse results due to competitive interactions between them andside effects. Furthermore, as the properties and concentration of thestored protein may change during its storage, the stabilizers mayexhibit unexpected side effects. Therefore, for a long-acting hGHconjugate, it is difficult to find a composition suitable for astabilizer capable of stabilizing both the physiologically activepeptide hGH and the immunoglobulin Fc region simultaneously. Inaddition, for a lyophilized formulation, the methods of lyophilizationand reconstituting should be controlled in various ways to maintainprotein stability and activity upon reconstitution. These methods mayalso vary depending on the composition of the formulation and theprotein used thereof.

Additionally, when a lyophilized formulation comprises a protein at highconcentration, the protein may aggregate during lyophilization becauseof the high concentration, and its handling also becomes difficult.Therefore, a protein at high concentration had been conventionallyobtained by preparing a lyophilized formulation comprising the proteinat low concentration and then reconstituting it with a small volumeinstead of performing lyophilization followed by reconstitution of thehigh-concentration protein. However, if the protein is reconstitutedwith a small volume, not only the protein but also other ingredientsincluded therein become too concentrated and too hypertonic for theformulation to be directly applicable to patients. Accordingly, there isa need for the development of a formulation that allows lyophilizing ofthe high-concentration protein as it is.

Recently, formulations of proteins and peptides that can be usedrepeatedly for the patients' convenience have been developed. However,these multi-use formulations should contain a preservative to preventmicrobial contamination after repeated administration and prior todisposal. The multi-use formulation containing a preservative has a fewadvantages over a single-use formulation. For example, as for thesingle-use formulation, a large amount of drug may be wasted dependingon the dosage, which may be reduced when the multi-use formulation isused. Furthermore, the multi-use formulation can be used several timeswithout the concern about microbial growth during a given time periodand, since it can be supplied in a single container, packaging can beminimized, leading to economic benefits. However, use of thepreservative may affect the protein stability. The most well-knownproblem associated with the use of a preservative is formation ofprecipitates. Precipitation of the protein may reduce the therapeuticeffect of the drug and induce an unexpected immune response whenadministered to the body. Therefore, it is critical to select anappropriate type and concentration of the preservative that maintain theability of microbial contamination without affecting the proteinstability.

In general, a formulation in solution state is developed in a syringeform. The most commonly-used type is a prefilled syringe, and a moreconvenient autoinjector is also frequently used. In addition, a peninjector which allows automated injection of a required dosage to apatient is used mainly for growth hormone, insulin, etc. Although theseinjection devices are convenient for administration of formulations insolution state, they cannot be used for the drugs which must belyophilized because of low stability.

In general, a lyophilized formulation is prepared in a reinforced glassvial separately from a solvent for dissolution. The two are mixed todissolve the lyophilized formulation immediately prior to injectionusing a syringe. The recent trend is from a lyophilized vial (e.g., areinforced glass vial) toward a single-use or multi-use syringe for thepatients' convenience. Examples include the dual chamber cartridge ofVetter (Germany).

DISCLOSURE Technical Problem

Under this background, the inventors of the present invention have madeefforts to develop a lyophilized formulation capable of maintaining thestability of a long-acting human growth hormone conjugate during thelyophilization process and capable of storing it for a long period oftime and a liquid formulation capable of stably storing a long-actinghuman growth hormone conjugate. As a result, they have found that when astabilizer comprising a buffer, a sugar alcohol and a non-ionicsurfactant is used, the stability of a long-acting hGH conjugate isincreased during lyophilization and storage, and thus a cost-effectiveand stable liquid formulation could be prepared. Also, it was confirmedthat when the concentration of the long-acting hGH conjugate is 10-58.5mg/mL, a sodium chloride-free liquid formulation with superior stabilitycan be provided. Furthermore, it was confirmed that the lyophilizedformulation of the present invention is not only stable during storageand transportation but also it has appropriate osmotic pressure andstability for subcutaneous injection when reconstituted. In addition, itwas confirmed that the lyophilized formulation can be used as amulti-use formulation since it maintains stability even when apreservative is included.

Technical Solution

The present invention is directed to providing a formulation of along-acting human growth hormone conjugate, comprising a long-actinghuman growth hormone (hGH) conjugate in which the human growth hormone(hGH) as a physiologically active peptide is linked to an immunoglobulinFc region, a buffer, a non-ionic surfactant and a sugar alcohol.

The present invention is also directed to providing a lyophilizedformulation of a long-acting hGH conjugate, comprising a lyophilizedmixture of an aqueous solution comprising a long-acting human growthhormone conjugate in which the hGH as a physiologically active peptideis linked to an immunoglobulin Fc region and an albumin-free solutioncomprising a buffer, a non-ionic surfactant and a sugar alcohol.

The present invention is also directed to providing a liquid formulationof a long-acting hGH conjugate, comprising a pharmaceutically effectiveamount of a long-acting hGH conjugate in which the hGH as aphysiologically active peptide is linked to an immunoglobulin Fc regionand an albumin-free stabilizer, wherein the stabilizer comprises abuffer, a non-ionic surfactant and a sugar alcohol.

The present invention is also directed to providing a method forpreparing the formulations.

The present invention is also directed to providing a method forreconstituting the lyophilized formulation, comprising adding a solutionfor reconstitution to the lyophilized mixture of an aqueous solutioncomprising a long-acting human growth hormone conjugate in which thehuman growth hormone as a physiologically active peptide is linked to animmunoglobulin Fc region and an albumin-free solution comprising abuffer, a non-ionic surfactant and a sugar alcohol.

The present invention is also directed to providing a kit comprising thelyophilized formulation of a long-acting hGH conjugate.

Advantageous Effects

Since the formulation of a long-acting hGH conjugate of the presentinvention does not comprises human serum albumin or any potentiallyhazardous factors, there is no concern of viral contamination. Inaddition, the formulation allows for a high stability of the long-actinghGH conjugate which is prepared by linking the hGH polypeptide to theimmunoglobulin Fc region, thus having a larger molecular weight whencompared to a wild-type and increased in vivo durability. In particular,the lyophilized formulation provides superior stability not only duringlyophilization but also after reconstitution, and also maintainsstability even when it contains a preservative, thus being useful as aformulation for multiple administrations.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a temperature gradient used in a lyophilization process ofthe present invention.

FIG. 2 shows a temperature gradient used in a lyophilization process ofthe present invention wherein primary drying is divided into two stages.

FIG. 3 shows a Vetter's dual chamber cartridge.

BEST MODE

In an aspect, the present invention provides a formulation of along-acting human growth hormone conjugate, comprising a long-actinghuman growth hormone (hGH) conjugate in which the human growth hormone(hGH) as a physiologically active peptide is linked to an immunoglobulinFc region, a buffer, a non-ionic surfactant and a sugar alcohol.

In another aspect, the present invention provides a lyophilizedformulation of a long-acting human growth hormone conjugate, comprisinga lyophilized mixture of an aqueous solution comprising a long-actinghGH conjugate in which the hGH as a physiologically active peptide islinked to an immunoglobulin Fc region and an albumin-free solutioncomprising a buffer, a non-ionic surfactant and a sugar alcohol.

In an exemplary embodiment, the buffer is an acetate buffer, a histidinebuffer or a citrate buffer.

In another exemplary embodiment, the buffer is an acetate buffer.

In another exemplary embodiment, the acetate is sodium acetate and thecitrate is sodium citrate.

In another exemplary embodiment, the pH of the buffer ranges from 5.0 to6.0.

In another exemplary embodiment, the sugar alcohol is mannitol orsorbitol.

In another exemplary embodiment, the sugar alcohol is included with aconcentration ranging from 1% (w/v) to 10% (w/v) of the total volume ofthe aqueous solution.

In another exemplary embodiment, the sugar alcohol is included with aconcentration ranging from 2.5% (w/v) to 5% (w/v).

In another exemplary embodiment, the non-ionic surfactant is polysorbate80.

In another exemplary embodiment, the concentration of the non-ionicsurfactant ranges from 0.001% (w/v) to 0.05% (w/v) of the total volumeof the aqueous solution.

In another exemplary embodiment, the albumin-free solution furthercomprises at least one selected from the group consisting of a sugar, apolyhydric alcohol and an amino acid.

In another exemplary embodiment, the amino acid is histidine or glycine.

In another exemplary embodiment, the concentration of the histidineranges from 1 to 10 mM.

In another exemplary embodiment, the concentration of the long-actinghGH conjugate ranges from 10 to 100 mg/mL.

In another exemplary embodiment, the albumin-free solution furthercomprises an isotonic agent.

In another exemplary embodiment, the isotonic agent is sodium chloride.

In another exemplary embodiment, the concentration of the sodiumchloride ranges from 0 to 200 mM.

In another exemplary embodiment, a container of the lyophilizedformulation is a vial, a dual chamber cartridge or a dual chambersyringe.

In another aspect, the present invention provides a liquid formulationof a long-acting hGH conjugate, comprising a pharmaceutically effectiveamount of a long-acting hGH conjugate in which the hGH as aphysiologically active peptide is linked to an immunoglobulin Fc regionand an albumin-free stabilizer, wherein the stabilizer comprises abuffer, a non-ionic surfactant and a sugar alcohol.

In an exemplary embodiment, the liquid formulation does not comprise anisotonic agent.

In another exemplary embodiment, the buffer is a citrate buffer, anacetate buffer or a histidine buffer.

In another exemplary embodiment, the sugar alcohol is mannitol orsorbitol.

In another exemplary embodiment, the sugar alcohol is included with aconcentration ranging from 2% (w/v) to 4.5% (w/v).

In another exemplary embodiment, the sugar alcohol is included with aconcentration of 4% (w/v).

In another exemplary embodiment, the pH of the buffer ranges from 5.0 to6.0.

In another exemplary embodiment, the pH of the buffer is 5.2.

In another exemplary embodiment, the non-ionic surfactant is polysorbate80.

In another exemplary embodiment, the concentration of the non-ionicsurfactant ranges from 0.001% (w/v) to 0.05% (w/v) of the total volumeof the formulation.

In another exemplary embodiment, the long-acting hGH conjugate isincluded in the formulation with a concentration ranging from 5.0 mg/mLto 60.0 mg/mL.

In another exemplary embodiment, the hGH has the same amino acidsequence as that of the wild-type hGH.

In another exemplary embodiment, the immunoglobulin Fc region is an Fcregion derived from IgG, IgA, IgD, IgE or IgM.

In another exemplary embodiment, each domain of the immunoglobulin Fcregion is a hybrid of domains with different origin derived from animmunoglobulin selected from the group consisting of IgG, IgA, IgD, IgEand IgM.

In another exemplary embodiment, the immunoglobulin Fc region is a dimeror a multimer composed of a single-chain immunoglobulin consisting ofdomains with the same origin.

In another exemplary embodiment, the immunoglobulin Fc region is an IgG4Fc region.

In another exemplary embodiment, the immunoglobulin Fc region is anaglycosylated human IgG4 Fc region.

In another exemplary embodiment, the conjugate is in such a form thatthe hGH is linked to the immunoglobulin Fc via a non-peptidyl polymer asa linker or via genetic recombination.

In another exemplary embodiment, the non-peptidyl polymer is selectedfrom the group consisting of a biodegradable polymer such aspolyethylene glycol, polypropylene glycol, a copolymer of ethyleneglycol and propylene glycol, a polyoxyethylated polyol, polyvinylalcohol, a polysaccharide, dextran, polyvinyl ethyl ether, polylacticacid (PLA) and polylactic-glycolic acid (PLGA), a lipid polymer, chitin,hyaluronic acid and a combination thereof.

In another exemplary embodiment, the non-peptidyl polymer ispolyethylene glycol.

In another exemplary embodiment, the formulation is for the treatment ofpituitary dwarfism, growth hormone deficiency, Prader-Willi syndrome oridiopathic short stature.

In another aspect, the present invention provides a lyophilizedformulation of a long-acting hGHconjugate, comprising a lyophilizedmixture of an aqueous solution comprising a long-acting hGH conjugate inwhich the hGH as a physiologically active peptide is linked to animmunoglobulin Fc region and an albumin-free solution comprising anacetate buffer, polysorbate 80 and mannitol.

In another aspect, the present invention provides a liquid formulationof a long-acting hGHconjugate, comprising a pharmaceutically effectiveamount of a long-acting hGH conjugate in which the hGHas aphysiologically active peptide is linked to an immunoglobulin Fc regionand an albumin-free stabilizer comprising a citrate buffer, polysorbate80 and mannitol, the stabilizer not comprising an isotonic agent.

In another aspect, the present invention provides a method for preparingthe lyophilized formulation, comprising lyophilizing a long-acting hGHconjugate in which the human growth hormone as a physiologically activepeptide is linked to an immunoglobulin Fc region and an albumin-freesolution comprising a buffer, a non-ionic surfactant and a sugaralcohol.

In another aspect, the present invention provides a method forreconstituting the lyophilized formulation, comprising adding a solutionfor reconstitution to the lyophilized mixture of an aqueous solutioncomprising a long-acting hGH conjugate in which the human growth hormone(hGH) as a physiologically active peptide is linked to an immunoglobulinFc region and an albumin-free solution comprising a buffer, a non-ionicsurfactant and a sugar alcohol included in the lyophilized formulation.

In an exemplary embodiment, the solution for reconstitution is water forinjection.

In another exemplary embodiment, the solution for reconstitution furthercomprises a preservative.

In another exemplary embodiment, the preservative is benzyl alcohol,m-cresol or phenol.

In another exemplary embodiment, the formulation reconstituted by themethod comprises the long-acting hGH conjugate with a concentrationranging from 10 to 100 mg/mL.

In another aspect, the present invention provides a kit comprising thelyophilized formulation of a long-acting hGH conjugate.

MODE FOR INVENTION

In an aspect, the present invention provides a formulation of along-acting human growth hormone conjugate, comprising a long-actinghuman growth hormone (hGH) conjugate in which the hGH as aphysiologically active peptide is linked to an immunoglobulin Fc region,a buffer, a non-ionic surfactant and a sugar alcohol.

As used herein, the term “long-acting human growth hormone (hGH)conjugate” refers to a conjugate in which the physiologically activepeptide human growth hormone is linked to an immunoglobulin Fc regionand the physiological activity of which has an increased in vivoduration when compared to a wild-type hGH. The term “long-acting” asused herein means that the physiological activity has a longer durationthan a wild-type hGH. As used herein, the term “conjugate” refers to aform in which the human growth hormone is coupled to an immunoglobulinFc region.

Specifically, the formulation may be a lyophilized formulation of along-acting hGH conjugate, comprising a lyophilized mixture of anaqueous solution comprising a long-acting hGH conjugate in which the hGHas a physiologically active peptide is linked to an immunoglobulin Fcregion and an albumin-free solution comprising a buffer, a non-ionicsurfactant and a sugar alcohol.

As used herein, the term “lyophilized formulation of a long-acting hGHconjugate” refers to a lyophilized formulation comprising a long-actinghGH conjugate. It includes a formulation comprising materials existingin solid state, obtained by lyophilizing a long-acting hGH conjugate anda substance to stabilize the same such as an excipient. In the presentinvention, the lyophilized formulation includes the lyophilizedsubstance itself. The lyophilized substance may also be referred to as alyophilized cake.

The lyophilized formulation is prepared by a lyophilization process ofsublimating water from a preparatory formulation comprising along-acting hGH conjugate and an excipient for stabilizing thelong-acting hGH conjugate. In the present invention, the lyophilizedformulation of a long-acting hGH conjugate may comprise atherapeutically effective amount of a long-acting hGH conjugate and atherapeutically effective amount of the hGH may be contained in asingle-use container or a multi-use container, although being notlimited thereto.

The lyophilized formulation may be contained in a vial (e.g., areinforced glass vial), a dual chamber cartridge or a dual chambersyringe, although being not limited thereto.

The lyophilized formulation of the present invention has a compositioncapable of stabilizing the long-acting hGH conjugate during alyophilization process and capable of maintaining the stability of theformulation when it is reconstituted after storage. In particular, thelyophilized formulation of the present invention is capable of providingstability even when the long-acting hGH conjugate is included with ahigh concentration ranging from 10 mg/mL to 100 mg/mL.

The lyophilized formulation of a long-acting hGH conjugate may be storedin a container and reconstituted when administration to a subject isnecessary.

As used herein, the term “reconstitution” means that the lyophilizedsubstance in solid state liquefied to allow for administration of thehGH conjugate. The concentration of the long-acting hGH conjugateincluded in the lyophilized formulation of the present invention rangesfrom 1 mg/mL to 150 mg/mL, specifically from 10 mg/mL to 120 mg/mL, morespecifically from 10 mg/mL to 100 mg/mL, upon reconstitution, althoughnot being limited thereto.

The reconstitution may be conducted by dissolving the lyophilizedsubstance by adding a solvent to a vial containing the lyophilizedsubstance or by adding a solvent to the lyophilized substance containedin a single-use syringe or a multi-use syringe, but is not speciallylimited thereto.

The lyophilized formulation of a long-acting hGH conjugate of thepresent invention is advantageous over the existing liquid formulationin that the hGH conjugate can be stably stored and the effectiveconcentration of the conjugate can be controlled. The concentrationafter the reconstitution may be identical to or different from theconcentration of the pre-lyophilized formulation during thelyophilization process.

The lyophilized formulation of the present invention comprises alyophilized mixture of an aqueous solution comprising a long-actinghGHconjugate and an albumin-free solution comprising a buffer, anon-ionic surfactant and a sugar alcohol.

As used herein, the term “albumin-free solution” refers to a substancewhich is capable of allowing the long-acting hGH conjugate to be stablystored and maintain its stability during lyophilizing and reconstitutionprocesses. In particular, the albumin-free solution refers to an aqueoussolution which comprises a long-acting hGH conjugate and an excipientstabilizing the same and, thus, provides stability of the long-actinghGH conjugate during a lyophilization process and allows the preparationof a lyophilized formulation having storage stability. Specifically, theaqueous solution comprises a buffer, a sugar alcohol and a non-ionicsurfactant. Further, an isotonic agent may be included for adjustment ofosmotic pressure. For proteins such as the long-acting hGH conjugate,storage stability is important not only to ensure an accurateadministration dosage but also to suppress the potential formation ofantigenic substances against the long-acting hGH conjugate. In thepresent invention, the term albumin-free solution may be usedinterchangeably with a “preformulation”.

Since the concentration of the long-acting hGH conjugate can becontrolled by adjusting the volume of the solution for reconstitutionadded to the lyophilized formulation, the concentration of thelong-acting hGH conjugate in the albumin-free solution is notparticularly limited. However, the formulation of the present inventionis advantageous in that even an albumin-free solution comprising thelong-acting hGH conjugate with a high concentration of 10-100 mg/mL orabove can be stably lyophilized, the prepared lyophilized formulationcan be quickly dissolved within 3 minutes and the stability of thelong-acting hGH conjugate can be maintained in the reconstitutedsolution.

The aqueous solution does not contain human serum albumin. Since thehuman serum albumin that can be used as a protein stabilizer is producedfrom human serum, there is a risk of contamination by pathogenic virusesderived from human. In addition, gelatin or bovine serum albumin maycause diseases or may induce an allergic response in some patients.Since the albumin-free solution of the present invention proteins doesnot contain heterologous proteins such as serum albumin derived fromhuman or animal or purified gelatin, there is no risk of viralinfection.

As used herein, the term “buffer” refers to a solution that is comprisedin the albumin-free solution of the present invention and works tomaintain a stable pH level of the formulation after a lyophilization orreconstitution process such that a sharp change in pH of the formulationis prevented to keep the activity of the long-acting hGH conjugatestable. The buffer may include an alkaline salt (e.g., sodium orpotassium phosphate, or monobasic or dibasic salts thereof), a citrate(e.g., sodium citrate or citric acid), an acetate (e.g., sodium acetateor acetic acid), histidine, any other pharmaceutically acceptable pHbuffering agent known in the art, or a combination thereof. The buffermay be specifically an acetate buffer, a histidine buffer or a citratebuffer, more specifically an acetate buffer or a citrate buffer,although not being limited thereto.

The concentration of the citrate or acetate that constitutes the bufferis specifically in a range from 5 to 100 mM, more specifically in arange from 10 to 50 mM, although not being limited thereto. The pH ofthe buffer is specifically in a range from 4.0 to 7.0, more specificallyin a range from 5.0 to 6.0, further more specifically in a range from5.2 to 6.0, although not being limited thereto.

As used herein, the term “sugar alcohol” refers to a hydrogenatedcarbohydrate that is comprised in the lyophilized formulation of thepresent invention and works to protect the protein of the long-actinghGH conjugate during a lyophilization process and to improve thestability of the long-acting hGH conjugate after reconstitution. Theconcentration of the sugar alcohol used in the present invention may bein a range from 1 to 10% (w/v) of the total volume of the formulation,although not being limited thereto. Specifically, the concentration ofthe sugar alcohol may be in a range from 2.5 to 5% (w/v). When theconcentration of the sugar alcohol is within this range, a reconstitutedformulation obtained by reconstitution using a solution forreconstitution of the same volume as that of the preformulation may havean osmotic pressure corresponding to that of an isotonic solution,although not being limited thereto.

The sugar alcohol used in the present invention may be at least oneselected from the group consisting of mannitol and sorbitol,specifically mannitol, but is not specially limited thereto. Beingincluded in the formulation of the present invention, the sugar alcoholsuch as mannitol may serve to adjust osmotic pressure. Specifically, theformulation obtained by reconstituting the lyophilized formulation ofthe present invention may be isotonic. However, it a hypertonic orhypotonic formulation is also suitable in the present invention.

As used herein, the term “non-ionic surfactant” refers to a substancethat reduces the surface tension of a protein solution to prevent theprotein from being adsorbed onto a hydrophobic surface or fromaggregating after reconstitution. Specific examples of the non-ionicsurfactant that can be used in the present invention include apolysorbate-type non-ionic surfactant, a poloxamer-type non-ionicsurfactant and a combination thereof. More specifically, apolysorbate-type non-ionic surfactant may be used. Examples of thepolysorbate-type non-ionic surfactant include polysorbate 20,polysorbate 40, polysorbate 60 and polysorbate 80, and among thempolysorbate 80 is preferred, although not being limited thereto. It maynot be appropriate to add the non-ionic surfactant at a highconcentration to the formulation, since a non-ionic surfactant at highconcentration can cause interference effects when protein is analyzed todetermine protein concentration or stability through analytic methodssuch as UV-spectrometric method or isoelectric focusing (IEF) and thusmake it hard to determine the protein stability accurately. Therefore,the lyophilized formulation of the present invention may comprise thenon-ionic surfactant specifically at a low concentration of 0.1% (w/v)or less, more specifically in a range from 0.001 to 0.1% (w/v), andfurther more specifically in a range from 0.001 to 0.05% (w/v), althoughnot being limited thereto.

Specifically, the albumin-free solution may further comprise at leastone selected from the group consisting of a sugar, a polyhydric alcoholand an amino acid. It was confirmed by the inventors of the presentinvention that, when histidine is further added as the amino acid,dissolution rate can be improved and reconstitution can be achievedwithout foaming.

Specific examples of the sugar that can be further included to increasethe storage stability of the long-acting hGH conjugate includemonosaccharides such as mannose, glucose, fucose and xylose andpolysaccharides such as lactose, maltose, sucrose, raffinose anddextran. Specific examples of the polyalcohol include propylene glycol,low-molecular-weight polyethylene glycol, glycerol, low-molecular-weightpolypropylene glycol and a combination thereof. Examples of the aminoacid include histidine or glycine, although not being limited thereto.The histidine, etc. may be present in the aqueous solution at aconcentration ranging from 1 to 10 mM, although not being limitedthereto.

The albumin-free solution may further comprise an isotonic agent forcontrol of osmotic pressure.

As used herein, the term “isotonic agent” refers to an agent thatmaintains an appropriate osmotic pressure when the long-acting hGHconjugate is administered into the body after being reconstituted. Theisotonic agent may have an effect of further stabilizing the long-actinghGH conjugate in solution. For example, the isotonic agent may be awater-soluble inorganic salt, specifically sodium chloride, although notbeing limited thereto. The concentration of sodium chloride used in thepresent invention is specifically in a range from 0 to 200 mM, althoughnot being limited thereto. Also, depending on the type and amount of thesubstances comprised in the formulation, the amount of the isotonicagent included can be adjusted such that the solution formulationcomprising all of the ingredients becomes isotonic.

The albumin-free solution may be lyophilized after being diluted ½-fold,¼-fold or further. It was confirmed by the inventors of the presentinvention that dissolution time can be reduced when the albumin-freesolution is lyophilized after being diluted ½-fold or ¼-fold (TestExample 1-(5)).

The lyophilized formulation of the present invention comprising thelyophilized mixture exhibits superior dissolution time. The dissolutiontime is one of important properties of a lyophilized substance. If thedissolution time is long, the protein has to be exposed to aconcentrated solution for a long time, during which it can be denatured.Also, since the incompletely dissolved product cannot be administered, ashort dissolution time can provide convenience for both the patients andthe physicians. However, the dissolution time is inevitably increased asthe protein concentration increases. Accordingly, development of aformulation with short dissolution time can be an important issue for ahigh-concentration lyophilized formulation.

It was confirmed by the inventors of the present invention that thelyophilized formulation of the present invention is dissolved within asshort as 10 seconds and as long as 3 minutes even when it comprised thelong-acting hGH conjugate at a high concentration of 10 mg/mL or above.

The desired dosage of a lyophilized product may be achieved bylyophilizing the target protein with a desired concentration and thenreconstituting the same with a volume of a preformulation, although notbeing specially limited thereto. Alternatively, the preformulation maybe lyophilized with an increased volume through dilution and thenreconstituted using a solution for reconstitution with a smaller volume.However, if the preformulation is overly diluted, the lyophilizationcycle (in particular, primary drying time) may be prolonged and thusproduction cost may be increased. Accordingly, the formulation of thepresent invention is also advantageous in terms of cost in that even thehigh-concentration long-acting hGH conjugate can be lyophilized withoutexcessive dilution.

In addition, the lyophilized formulation of the present invention mayfurther comprise other ingredients or materials that are known in theart in addition to the above-described buffer, isotonic agent, sugaralcohol, non-ionic surfactants and the preservative included in thesolution for reconstitution, unless they do not diminish the effect ofthe present invention.

The inventors of the present invention prepared, as a preformulation, alyophilized formulation comprising a long-acting hGH conjugate and analbumin-free solution comprising a buffer, a sugar alcohol and asurfactant and evaluated its stability and dissolution rate.Specifically, a long-acting hGH conjugate was lyophilized with aconcentration of 19.5 mg/mL or 78.0 mg/mL using a pre-lyophilizedformulation comprising a 20 mM citrate buffer of pH 5.2 or pH 5.6, 150mM sodium chloride, 5% mannitol and 0.005% polysorbate 80 and thenreconstituted using distilled water. Superior stability was superior atthe above concentrations. In particular, the stability was better at thehigher concentration of 78.0 mg/mL. Also, good stability was achieved atpH 5.2 and 5.6 both (Test Example 1-(1)). In addition, when along-acting hGH conjugate was dissolved in a pre-lyophilized formulationcomprising a 20 mM acetate buffer of pH 5.2 or pH 5.6, 150 mM NaCl, 5%mannitol and 0.005% polysorbate 80 and then reconstituted using asolution for reconstitution comprising m-cresol, benzyl alcohol orphenol as a preservative, the stability was maintained. This resultconfirms that a reconstituted formulation with preserved activity can beprepared by reconstituting a lyophilized substance with a solution forreconstitution comprising a preservative. In particular, when theaqueous acetate solution was used, superior stability could be achievedwithout precipitation even when the reconstitution was conducted using asolution for reconstitution comprising a preservative (Test Example1-(2)). Furthermore, it was confirmed that the dissolution time of theprepared lyophilized formulation is increased as the concentration ofthe conjugate increases from 19.5 mg/mL to 39.0 mg/mL to 58.5 mg/mL andto 70.0 mg/mL (Test Example 1-(3)). In addition, superior dissolutiontime and stability were achieved even when the albumin-free solution didnot contain a salt and the dissolution rate could be increased byincreasing the concentration of mannitol. And, when histidine was added,it was possible to reduce the dissolution time without increasing theconcentration of mannitol (Test Example 1-(4)). Further, the inventorsfound out that the dissolution rate is further improved when thepreformulation is diluted to decrease density (Test Example 1-(5)) andestablished the optimized drying condition under which the lyophilizedsubstance can be completely dried (Test Example 1-(6)). It was alsodemonstrated that the presence of mannitol greatly affects thedissolution time (Test Example 1-(7)) and that an isotonic osmoticpressure can be achieved with a dissolution time of 30 seconds or lesswhen the mannitol is concentration 4-4.5% even for thehigh-concentration long-acting hGH conjugate of 58.5 mg/mL (Test Example1-(8)). In addition, the stability was maintained nearly constant evenafter the prepared lyophilized formulations were stored at 4° C. or 25°C. for 6 months. Also, the stability was maintained even after thelyophilized formulations were stored at 25° C. for 2 weeks afterreconstitution (Test Example 1-(9)).

Specifically, the formulation may be a liquid formulation of along-acting hGH conjugate, comprising a long-acting hGH conjugate inwhich the hGH as a physiologically active peptide is linked to animmunoglobulin Fc region and an albumin-free stabilizer, wherein thestabilizer comprises a buffer, a non-ionic surfactant and a sugaralcohol.

The long-acting hGH conjugate is the same as described above.

As used herein, the term “liquid formulation of a long-acting hGHconjugate” refers to a liquid formulation which comprises a long-actinghGH conjugate. The liquid formulation includes liquid formulations forboth internal and external application. In the present invention, theliquid formulation of a long-acting hGH conjugate may comprise apharmaceutically effective amount of the long-acting hGH conjugate. Ingeneral, the pharmaceutically effective amount of hGH corresponds toabout 1 to 3 mg in a single-use vial, but is not limited thereto.

And, the concentration of the long-acting hGH conjugate comprised in theliquid formulation of the present invention ranges specifically from 5.0to 60.0 mg/mL, although not being limited thereto.

The liquid formulation of a long-acting hGH conjugate comprises apharmaceutically effective amount of a long-acting hGH conjugate and analbumin-free stabilizer.

As used herein, the term “stabilizer” refers to a substance that allowsthe long-acting hGH conjugate to be stored stably. Specifically, thestabilizer comprises a buffer, a sugar alcohol and a non-ionicsurfactant. With regard to proteins such as the long-acting hGHconjugate, the storage stability is important for ensuring dose accuracyand suppressing the formation of potential antigens against thelong-acting hGH conjugate.

As used herein, the term “buffer” refers to a solution that is comprisedin the stabilizer of the present invention and works to maintain astable pH level of the formulation, thereby preventing a drastic changein pH to maintain the activity of the long-acting hGH conjugate stable.The description of the buffer stated above with regard to thelyophilized formulation also applies here.

The buffer may be a citrate buffer, an acetate buffer or a histidinebuffer, specifically a citrate buffer, although not being limitedthereto. The concentration of the salt that constitutes the buffer isspecifically in a range from 5 to 100 mM, more specifically in a rangefrom 10 to 50 mM, although not being limited thereto. The pH of thebuffer is in a range from 4.0 to 7.0, more specifically in a range from5.0 to 6.0, further more specifically in a range from 5.2 to 6.0, mostspecifically 5.2, although not being limited thereto.

As used herein, the term “sugar alcohol” refers to a hydrogenatedcarbohydrate that is comprised in the liquid formulation of the presentinvention and works to improve the stability of the long-acting hGHconjugate. The concentration of the sugar alcohol used in the presentinvention is specifically in a range from 1 to 10% (w/v) of a totalvolume of the formulation, and more specifically in a range from 2%(w/v) to 4.5% (w/v), further more specifically 4% (w/v), although notbeing limited thereto. The sugar alcohol used in the present inventionmay be at least one selected from the group consisting of mannitol andsorbitol, specifically mannitol, although not specially limited thereto.

As used herein, the term “non-ionic surfactant” refers to a substancethat reduces the surface tension of a protein solution to prevent theprotein from being adsorbed onto a hydrophobic surface or fromaggregating. Specific examples of the non-ionic surfactant that can beused in the present invention are the same as described above.

The formulation of the present invention may be one not containing anisotonic agent.

The isotonic agent is the same as described above.

The inventors of the present invention found that a formulationcomprising an aqueous acetate or citrate solution of pH 5.2 as a bufferand comprising 4% (w/v) mannitol but not comprising an isotonic agentexhibits superior stability. In particular, a formulation comprising anaqueous citrate solution of pH 5.2 as a buffer and comprising 4% (w/v)mannitol but not comprising an isotonic agent exhibited the higheststability at the long-acting hGH conjugate concentration of 10.0 mg/mL.

Specific examples of the sugar that can be further included to increasethe storage stability of the long-acting hGH conjugate are the same asdescribed above. In addition, the liquid formulation of the presentinvention may further comprise other ingredients or materials that areknown in the art in addition to the above-described buffer, sugaralcohol and non-ionic surfactants, unless they do not diminish theeffect of the present invention.

In particular, the liquid formulation of the present invention mayfurther comprise a preservative.

It was found that a formulation of a long-acting hGH conjugate notcontaining an isotonic agent exhibits the best stability of thelong-acting hGH conjugate in a buffer at pH 5.2 when it comprises 4%mannitol (w/v). In particular, the best stability was achieved at thelong-acting hGH conjugate concentration of 10.0 mg/mL when an acetatebuffer was used (Test Example 2).

The formulation of the present invention may be for the treatment ofpituitary dwarfism, growth hormone deficiency, Prader-Willi syndrome oridiopathic short stature and may be injected into after reconstitutionto treat the diseases.

Hereinafter, the long-acting hGH conjugate is described in detail.

As used herein, the term “human growth hormone (hGH)” refers to apeptide hormone that stimulates growth, cell reproduction andregeneration in humans. The information on the sequence of the hGH canbe obtained from common database such as the NCBI GenBank. In addition,the scope of the hGH in the present invention includes a proteinpossessing an amino acid sequence having a sequence homology of 70% orhigher, specifically 80% or higher, more specifically 90% or higher,even more specifically 95% or higher, and most specifically 98% orhigher to an amino acid sequence of a wild-type hGH, as long as it has ahGH activity. Also, as long as its biological activity is notsignificantly changed, any mutant derived from a wild-type hGH bysubstitution, deletion, or insertion of amino acid residues may be usedin the present invention.

The hGH useful in the present invention may have an amino acid sequenceof a wild-type hGH, its variant, its derivative, or fragments thereof.

As used herein, the term “hGH variant” refers to a peptide having atleast one amino acid sequences different from those of the wild-type hGHwhile demonstrating the hGH activity. The hGH variant may be prepared bysubstitution, addition, deletion, or modification of some amino acids ofthe wild-type hGH or a combination thereof.

As used herein, the term “hGH derivative” refers to a peptide having atleast 80% amino acid sequence homology to the wild-type hGH andexhibiting the hGH activity, in which some groups of the amino acidresidues are chemically substituted (e.g., alpha-methylation,alpha-hydroxylation), deleted (e.g., deamination), or modified (e.g.,N-methylation).

As used herein, the term “hGH fragment” refers to a peptide in which atleast one amino acids are added or deleted at the N-terminal or theC-terminal of the hGH while retaining the hGH activity. The added aminoacid be one which does not naturally occur (for example, D-amino acid).

In addition, the hGH used in the present invention may be obtained froma native or recombinant protein. Specifically, it is the recombinant hGHprepared by using E. coli as a host cell, although not being limitedthereto.

As used herein, the term “immunoglobulin Fc region” refers to a part ofimmunoglobulin excluding the variable regions of the heavy chain andlight chain, the heavy-chain constant region 1 (C_(H)1) and thelight-chain constant region 1 (C_(L)1) of the immunoglobulin. Theimmunoglobulin Fc region may be the heavy-chain constant region 2(C_(H)2) and the heavy-chain constant region 3 (C_(H)3) of animmunoglobulin, and may further comprise a hinge region at theheavy-chain constant region, although not being limited thereto. Also,the immunoglobulin Fc region of the present invention may be an extendedFc region that comprises a portion or full of the heavy-chain constantregion 1 (C_(H)1) and/or the light-chain constant region 1 (C_(L)1)except for the variable regions of the heavy chain and light chain ofimmunoglobulin, as long as it has substantially the same or improvedeffect as compared to the wild-type protein. Also, the immunoglobulin Fcregion may be a fragment wherein a considerably long portion of theamino acid sequence corresponding to C_(H)2 and/or C_(H)3 is deleted.That is, the immunoglobulin Fc region of the present invention maycomprise 1) a C_(H)1 domain, a C_(H)2 domain, a C_(H)3 domain and aC_(H)4 domain, 2) a C_(H)1 domain and a C_(H)2 domain, 3) a C_(H)1domain and a C_(H)3 domain, 4) a C_(H)2 domain and a C_(H)3 domain, 5) acombination of at least one domains and an immunoglobulin hinge region(or a portion of the hinge region), and 6) a dimer of a domain of theheavy-chain constant regions and a light-chain constant region, althoughnot being limited thereto.

The immunoglobulin Fc region of the present invention comprises a nativeamino acid sequence and an amino acid sequence derivative (mutant)thereof. The amino acid sequence derivative refers to the sequencehaving different sequence from the native sequence by deletion,insertion, non-conservative or conservative substitution of at least oneamino acid residues of the native amino acid sequence, or combinationsthereof. For example, in IgG Fc, amino acid residues at positions 214 to238, 297 to 299, 318 to 322, or 327 to 331 which are known to beimportant for protein binding may be suitable targets for modification.

Also, other types of derivatives may be used including the derivativeswherein a region capable of forming a disulfide bond is deleted, fewamino acid residues at the N-terminal of a native Fc are eliminated, ora methionine residue is added at the N-terminal of the native Fc.Further, in order to eliminate the function of effector, acomplement-binding site, for example C1q-binding site or antibodydependent cell mediated cytotoxicity (ADCC) site, may be deleted.Techniques for preparing such sequence derivatives of the immunoglobulinFc region are disclosed in WO 97/34631 and WO 96/32478.

Substitution of amino acids in proteins and peptides, which do notchange the overall protein activities, are known in the art (H. Neurath,R. L. Hill, The Proteins, Academic Press, New York, 1979). Themost-commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu,Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro,Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, in bothdirections. In some cases, the Fc region may be modified byphosphorylation, sulfation, acrylation, glycosylation, methylation,farnesylation, acetylation, amidation, and the like. The aforementionedFc derivatives demonstrate the same biological activity as the Fc regionof the present invention, and they have an enhanced structural stabilityagainst heat, pH, and the like.

In addition, these Fc regions may be obtained from native proteinsisolated from humans or other animals including cows, goats, swine,mice, rabbits, hamsters, rats and guinea pigs, or may be recombinantsobtained from transformed animal cells or microorganisms or derivativesthereof. Here, the method of obtaining Fc regions from nativeimmunoglobulin may include isolating the whole immunoglobulins fromhuman or animal bodies and treating them with a protease. When papain isused for digesting immunoglobulins, they are cleaved into Fab and Fcregions, and when pepsin is used, the immunoglobulin is cleaved intopF′c and F(ab)₂. These fragments may be separated by size exclusionchromatography to isolate Fc or pF′c. Specifically, a human-derived Fcregion is a recombinant immunoglobulin Fc region obtained from amicroorganism.

In addition, the immunoglobulin Fc region of the present invention maybe in a form of native sugar chains, longer sugar chains than nativeform, shorter sugar chains than native form, or a deglycosylated form.The extension or removal of the immunoglobulin Fc sugar chains may bedone by using common methods in the art including chemical methods,enzymatic methods, and gene engineering method using a microorganism.The removal of sugar chains from an immunoglobulin Fc region results ina drastic decrease in its binding affinity to C1q of the firstcomplement component C1 and thus antibody-dependent cell-mediatedcytotoxicity or complement-dependent cytotoxicity is reduced or removed,and the occurrence of unnecessary immune responses in vivo can beavoided. In this regard, a deglycosylated or aglycosylatedimmunoglobulin Fc region is more preferable form as a drug carrier forthe object of the present invention.

In addition, the immunoglobulin Fc region may be one derived from IgG,IgA, IgD, IgE and IgM, or those prepared by a combination or hybridthereof. Specifically, it is derived from IgG or IgM, which are amongthe most abundant proteins in human blood, and most specifically fromIgG, which is known to enhance the half-life of a ligand-bindingprotein. The immunoglobulin Fc may be generated by treating a native IgGwith a certain protease, or by transformed cells using the geneticrecombination technique. Specifically, the immunoglobulin Fc is arecombinant human immunoglobulin Fc produced in E. coli.

Meanwhile, the term “combination”, as used herein, refers to aconjugation between a polypeptide encoding single-chain immunoglobulinFc regions of the same origin and a single-chain polypeptide ofdifferent origin when forming a dimer or multimer. That is, a dimer ormultimer can be formed from two or more fragments selected from thegroup consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragments.

As used herein, the term “hybrid” refers to the presence of at least twosequences corresponding to immunoglobulin Fc fragments of differentorigins in a single-chain immunoglobulin Fc region. In the presentinvention, various types of hybrids may be used. That is, a hybrid ofdomains may be composed of one to four domains selected from the groupconsisting of C_(H)1, C_(H)2, C_(H)3 and C_(H)4 of IgG Fc, IgM Fc, IgAFc, IgE Fc and IgD Fc, and may comprise a hinge region.

Meanwhile, IgG may also be divided into subclasses, IgG1, IgG2, IgG3 andIgG4, and a combination or hybrid thereof is also possible in thepresent invention, specifically IgG2 and IgG4 subclasses, and mostspecifically Fc region of IgG4 that lacks an effector function such ascomplement-dependent cytotoxicity. In other words, the most preferableimmunoglobulin Fc region of the conjugate in the present invention is ahuman IgG4-derived non-glycosylated Fc region. The human-derived Fcregion is preferred to a non-human derived Fc region which can act as anantigen in human body and cause undesirable immune responses such asproduction of new antibodies against the antigen.

The long-acting hGH conjugate of the present invention can be preparedby combining a hGH prepared from a native or recombinant form by anymethod and an immunoglobulin Fc region prepared by treating a wild-typeIgG with a certain protease or produced from a transformed cell by usingthe recombination technique.

As a combining method used for this purpose, the conjugate can beprepared by cross-linking the hGH and the immunoglobulin Fc region usinga non-peptidyl polymer or can be produced as a fusion protein whereinthe hGH and the immunoglobulin Fc region are linked using therecombination technique. That is, the conjugate can be produced in aform where the hGH and the immunoglobulin Fc are linked via anon-peptidyl linker, or in a form of a fusion protein of the hGH and theimmunoglobulin Fc. The fusion protein comprises a form where the hGH andthe immunoglobulin Fc are combined via a peptidyl linker, although notbeing limited thereto.

As used herein, the term “non-peptidyl polymer” refers to abiocompatible polymer in which two or more repeating units are combinedand the repeating units are connected to each other by any covalentbonding except for peptide bonding. In the present invention, the termnon-peptidyl polymer may be used interchangeably with the termnon-peptidyl linker.

The non-peptidyl polymer used for cross-linking may be selected from thegroup consisting of a biodegradable polymer including polyethyleneglycol, polypropylene glycol, an ethylene glycol-propylene glycolcopolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide,dextran, polyvinyl ethyl ether, polylactic acid (PLA) orpolylactic-glycolic acid (PLGA), a lipid polymer, chitin, hyaluronicacid and a combination thereof. Specifically, polyethylene glycol (PEG)may be used, although not being limited thereto. In addition, theirderivatives that are already known in the art and derivatives that canbe easily prepared by a method known in the art may be included in thescope of the present invention.

For preparation of the long-acting hGH conjugate of the presentinvention, references such as Korean Patent No. 0725315 are disclosed inthe present invention as cited references. Those skilled in the art canproduce the long-acting hGH conjugate of the present invention byconsulting the references, although not being limited thereto.

In another aspect, the present invention provides a lyophilizedformulation of a long-acting hGH conjugate, comprising a lyophilizedmixture of an aqueous solution comprising a long-acting hGH conjugate inwhich the hGH as a physiologically active peptide is linked to animmunoglobulin Fc region and an albumin-free solution comprising anacetate buffer, polysorbate 80 and mannitol.

The hGH, the immunoglobulin Fc region, the long-acting hGH conjugate,the albumin-free solution, the lyophilizing and the lyophilizedformulation are the same as descried above.

In another aspect, the present invention provides a liquid formulationof a long-acting hGHconjugate, comprising a pharmaceutically effectiveamount of a long-acting hGHconjugate in which the hGHas aphysiologically active peptide is linked to an immunoglobulin Fc regionand an albumin-free stabilizer comprising a citrate buffer, polysorbate80 and mannitol, the stabilizer not comprising an isotonic agent.

The hGH, the immunoglobulin Fc region, the long-acting hGHconjugate, thealbumin-free stabilizer and the liquid formulation are the same asdescried above.

In another aspect, the present invention provides a method for preparingthe lyophilized formulation, comprising lyophilizing a long-acting hGHconjugate and an albumin-free solution comprising a buffer, a non-ionicsurfactant and a sugar alcohol.

The long-acting hGH conjugate, the buffer, the non-ionic surfactant, thesugar alcohol, the albumin-free solution, the lyophilization and thelyophilized formulation are the same as descried above.

In another aspect, the present invention provides a method forreconstituting the lyophilized formulation, comprising adding a solutionfor reconstitution to the lyophilized formulation.

The lyophilized formulation and the reconstitution are the same asdescried above.

As used herein, the term “solution for reconstitution” refers to asolution which is added to a lyophilized substance in solid state toreconstitute the same. The solution for reconstitution may be water forinjection, e.g. sterilized distilled water, but is not specially limitedthereto.

Also, the solution for reconstitution may further comprise apreservative.

As used herein, the term “preservative” refers to a substance thatsubstantially reduces bacterial or fungal contamination in aformulation. Especially, it is comprised in the formulation tofacilitate the production of a formulation for multiple dosing. Examplesof preservative include octadecyl dimethyl benzyl ammonium chloride,hexamethonium chloride, benzalkonium chloride (mixture ofalkylbenzyldimethylammonium chloride which has a long alkyl chain), andbenzethonium chloride. Other types of preservatives include: aromaticalcohols such as phenol, butyl alcohol and benzyl alcohol; alkyl parabensuch as methylparaben or propylparaben; catechol; resorcinol;cyclohexanol; 3-pentanol; and m-cresol, but are not limited thereto. Thepreservative in the liquid formulation of the present invention isspecifically benzyl alcohol, m-cresol or phenol, more specificallybenzyl alcohol, although not being limited thereto. The concentration ofthe preservative is specifically in a range from 0.001 to 0.9% (w/v),more specifically in a range from 0.1 to 0.9% (w/v), although not beinglimited thereto.

The formulation of the present invention reconstituted as describedabove may contain the long-acting hGH conjugate with a concentrationranging from 10 to 100 mg/mL, although not being limited thereto.

In another aspect, the present invention provides a kit comprising thelyophilized formulation and a solution for reconstitution.

The lyophilized formulation is the same as described above.

The kit comprises the lyophilized formulation and a solution forreconstitution and may further comprise a composition, solution orapparatus comprised of at least one other ingredient suitable for thereconstitution.

Hereinafter, the present invention is described in more detail withreference to examples. However, these examples are for illustrativepurposes only, and the invention is not intended to be limited by theseexamples.

Preparation Example Preparation of Long-Acting Human Growth Hormone(hGH) Conjugate

ALD-PEG-ALD, which is a polyethylene glycol with a molecular weight ofabout 3.4 kDa having aldehyde groups at both ends, was conjugated withthe human growth hormone (hGH, molecular weight: 22 kDa), and thenlinked to the N-terminal of a human IgG4-derived aglycosylated Fc region(about kDa). Through this, the final product hGH-PEG-Fc conjugate(hereinafter, referred to as “long-acting hGH conjugate”) which is arepresentative long-acting hGH conjugate of the present invention wasprepared and purified.

Test Example 1 Evaluation of Lyophilized Formulation of Long-Acting hGHConjugate

(1) Analysis of Stability of Lyophilized Formulation of Long-Acting hGHConjugate Depending on Concentration and Buffer

After preparing lyophilized formulations comprising the long-acting hGHconjugate at concentrations described in Table 1, stability was analyzedafter reconstitution. The effect of buffer and pH on the stability ofthe long-acting hGH conjugate was also analyzed.

As described in Table 1, the long-acting hGH conjugate was lyophilizedwith the given concentration using a pre-lyophilized formulationcomprising a buffer, sodium chloride (NaCl), mannitol and polysorbate80, which was then reconstituted using distilled water. The lyophilizingconsisted of primary drying and secondary drying steps. The temperaturegradient of the lyophilizing was set as freezing followed by primarydrying (4° C.) and secondary drying (20° C.), as shown in FIG. 1. Thereconstitution was conducted by dissolving the lyophilized formulationwith distilled water of the same volume as that of the formulationbefore the lyophilization. The reconstituted liquid formulation wasstored at 40° C. for 4 weeks and the stability was analyzed by ionexchange chromatography (IE-HPLC). The result is shown in Table 2. InTable 2, IE-HPLC (%) indicates the purity of the long-acting hGHconjugate at the given time.

TABLE 1 Conc. Buffer Salt Sugar alcohol Surfactant Example 19.5 mg/mL 20mM 150 mM 5% mannitol 0.005% 1 sodium NaCl polysorbate (Ex. 1) citrate80 (pH 5.2) Ex. 2 78.0 mg/mL 20 mM 150 mM 5% mannitol 0.005% sodium NaClpolysorbate citrate 80 (pH 5.2) Ex. 3 78.0 mg/mL 20 mM 150 mM 5%mannitol 0.005% sodium NaCl polysorbate acetate 80 (pH 5.6)

TABLE 2 IE-HPLC (%) Week 0 Week 1 Week 2 Week 4 Example 1 96.2 93.6 87.176.8 Example 2 96.4 95.9 92.8 82.0 Example 3 96.4 93.6 88.8 82.2

As seen from Table 2, the liquid formulation of a long-acting hGHconjugate showed no difference in stability depending on concentrationafter being stored at 40° C. for 4 weeks. Accordingly, it was confirmedthat stability can be provided even to the long-acting hGH at highconcentration. Also, it was confirmed that the stability is maintainedwhen the buffer and the pH were changed (compare Examples 2 and 3).

(2) Analysis of Stability and Solubility of Lyophilized Formulation ofLong-Acting Human Growth Hormone (hGH) Conjugate Depending onPreservative

Using the formulations of Example 2 (20 mM sodium citrate, pH 5.2, 150mM sodium chloride, 5% mannitol, 0.005% polysorbate 80) and Example 3(20 mM sodium acetate, pH 5.6, 150 mM sodium chloride, 5% mannitol,0.005% polysorbate 80) of Test Example 1-(1) and an isotonic formulationprepared from the formulation of Example 3 (20 mM sodium acetate, pH5.6, 4% mannitol, 0.005% polysorbate 80), the long-acting hGH conjugatewas mixed at concentrations of 68.25 mg/mL and 58.5 mg/mL as describedin Table 3, which were then lyophilized. After reconstituting using thesolution for reconstitutions comprising preservatives described in Table3, dissolution time and stability were measured. The product state wascompared with unaided eyes. The lyophilizing and reconstitution wereconducted in the same manner as described in Test Example 1-(1). Thereconstituted liquid formulation was stored at 25° C. for 4 weeks andthen stability was evaluated by ion exchange chromatography (IE-HPLC)and visual inspection. The result is shown in Table 4. In Table 4,IE-HPLC (%) indicates the purity of the long-acting hGH conjugate at thegiven time.

TABLE 3 Sugar Surfac- Preser- Conc. Buffer Salt alcohol tant vative Ex.4 68.25 20 mM 150 mM 5% 0.005% 0.3% mg/mL sodium NaCl mannitol polysor-m-cresol citrate bate 80 (pH 5.2) Ex. 5 68.25 20 mM 150 mM 5% 0.005%0.9% mg/mL sodium NaCl mannitol polysor- benzyl citrate bate 80 alcohol(pH 5.2) Ex. 6 68.25 20 mM 150 mM 5% 0.005% 0.3% mg/mL sodium NaClmannitol polysor- m-cresol acetate bate 80 (pH 5.6) Ex. 7 68.25 20 mM150 mM 5% 0.005% 0.9% mg/mL sodium NaCl mannitol polysor- benzyl acetatebate 80 alcohol (pH 5.6) Ex. 8 58.5 20 mM — 4% 0.005% 0.3% mg/mL sodiummannitol polysor- m-cresol acetate bate 80 (pH 5.6) Ex. 9 58.5 20 mM —4% 0.005% 0.3% mg/mL sodium mannitol polysor- phenol acetate bate 80 (pH5.6)

TABLE 4 IE-HPLC (%) Week 0 Week 1 Week 2 Week 4 Remarks Example 4 96.793.2 90.0 83.7 Precipitation occurred on week 3 Example 5 96.7 92.6 89.883.1 Precipitation occurred on week 3 Example 6 96.6 93.1 89.4 87.7 —Example 7 96.5 93.1 89.6 87.5 — Example 8 97.6 95.4 92.3 88.1 — Example9 97.4 95.3 92.4 87.4 —

As seen from Table 4, the stability of the long-acting hGH conjugate wasmaintained better in Examples 6 and 7 than in Examples 4 and 5. However,as can be seen from the results for Examples 6-9, there was nodifference in the stability of the long-acting hGH conjugate dependingon the kind of the preservatives. But, when the solution forreconstitution containing m-cresol was used, the resulting liquidformulation was hazy during the dissolution.

(3) Analysis of Solubility of Lyophilized Formulation of Long-Acting hGHConjugate Depending on Conjugate Concentration

Lyophilized formulations comprising the long-acting hGH conjugate atdifferent concentrations were prepared and their product state andsolubility upon reconstitution were evaluated. Using the formulation ofExample 1 (20 mM sodium citrate, pH 5.2, 150 mM sodium chloride, 5%mannitol, 0.005% polysorbate 80) of Test Example 1-(1), the long-actinghGH conjugate was mixed at different concentrations as described inTable 5, which were then lyophilized. After reconstituting usingdistilled water, dissolution time was measured. The lyophilization andreconstitution were conducted in the same manner as described in TestExample 1-(1). The product state was compared with unaided eyes. Thereconstitution was performed using an auto shaker set to 60° and 30 rpm.The time required for complete dissolution is given in Table 6.

TABLE 5 Sugar Conc. Buffer Salt alcohol Surfactant Ex. 1 19.5 mg/mL 20mM sodium 150 mM 5% 0.005% citrate (pH 5.2) NaCl mannitol polysorbate 80Ex. 10 39.0 mg/mL 20 mM sodium 150 mM 5% 0.005% citrate (pH 5.2) NaClmannitol polysorbate 80 Ex. 11 58.5 mg/mL 20 mM sodium 150 mM 5% 0.005%citrate (pH 5.2) NaCl mannitol polysorbate 80 Ex. 12 70.0 mg/mL 20 mMsodium 150 mM 5% 0.005% citrate (pH 5.2) NaCl mannitol polysorbate 80

TABLE 6 Example 1 Example 10 Example 11 Example 12 Dissolution time 1030 90 150 (sec)

Although the product state of the lyophilized substance was stableregardless of concentration, more rigid cakes could be observed athigher concentrations. As seen from Table 6, the dissolution time wasincreased with concentration.

(4) Analysis of Stability and Solubility of Lyophilized Formulation ofLong-Acting Human Growth Hormone (hGH) Conjugate Depending on Stabilizer

Lyophilized formulations of the long-acting hGH conjugate were preparedusing different stabilizers and their dissolution time, dissolutionstate and long-acting hGH conjugate stability were evaluated.Preformulations were prepared with the compositions described in Table7, which were then used to freeze-dry the long-acting hGH conjugate at78.0 mg/mL. After reconstituting using distilled water, dissolution timewas measured. The lyophilization and reconstitution were conducted inthe same manner as described in Test Example 1-(1). The product statewas compared with unaided eyes. The reconstitution was performed usingan auto shaker set to 60° and 30 rpm. The time required for completedissolution is given in Table 8.

Also, after storing the reconstituted liquid formulation at 40° C. for 4weeks, stability was evaluated by ion exchange chromatography (IE-HPLC).In Table 9, IE-HPLC (%) indicates the residual rate of the long-actinghGH conjugate at the given time relative to the initial value.

TABLE 7 Sugar alcohol and other Conc. Buffer Salt stabilizer SurfactantEx. 13 78.0 20 mM sodium 150 mM   5% mannitol 0.005% mg/mL citrate (pH5.2) NaCl polysorbate 80 Ex. 14 78.0 20 mM sodium 150 mM   5% mannitol 0.02% mg/mL citrate (pH 5.2) NaCl polysorbate 80 Ex. 15 78.0 20 mMsodium 150 mM  10% mannitol 0.005% mg/mL citrate (pH 5.2) NaClpolysorbate 80 Ex. 16 78.0 20 mM sodium 150 mM 2.5% mannitol 0.005%mg/mL citrate (pH 5.2) NaCl polysorbate 80 Ex. 17 78.0 20 mM sodium  10%mannitol 0.005% mg/mL citrate (pH 5.2) polysorbate 80 Ex. 18 78.0 20 mMsodium   5% mannitol 0.005% mg/mL citrate (pH 5.2)   2% glycinepolysorbate 80 Ex. 19 78.0 20 mM sodium   5% mannitol 0.005% mg/mLcitrate (pH 5.2) 5 mM histidine polysorbate 80

TABLE 8 Ex. Ex. Ex. Ex. Ex. Ex. Ex. 13 14 15 16 17 18 19 Dissolution 120120 80 180 90 130 90 time (sec)

TABLE 9 IE-HPLC (%) Week 0 Week 2 Week 4 Example 13 100.0 97.8 87.9Example 14 100.0 97.8 88.3 Example 15 100.0 96.3 85.8 Example 16 100.098.1 86.8 Example 17 100.0 94.7 82.6 Example 18 100.0 96.3 81.6 Example19 100.0 95.4 83.7

As seen from Table 8, high dissolution rate was obtained when theconcentration of mannitol was high. Also, it was confirmed that theaddition of 5 mM histidine leads to improved dissolution rate. Theformulations of Examples 15 and 17 showed severe foaming duringreconstitution as compared to the formulation of Example 19. As seenfrom Table 9, the stability after the dissolution was similar for eachformulation. But, when sodium chloride was included as the stabilizer,the stability of the long-acting hGH conjugate was a little higher.

(5) Analysis of Solubility of Lyophilized Formulation Depending onDensity of Lyophilized Substance and Concentration of Long-Acting hGHConjugate

Using the formulation of Example 19 (20 mM sodium citrate, pH 5.2, 5%(w/v) mannitol, 5 mM histidine, 0.005% (w/v) polysorbate 80) of TestExample 1-(4), the solubility of the lyophilized substance depending onthe long-acting hGH conjugate concentration was analyzed.Preformulations were prepared with the compositions described in Table10 and then lyophilized. During the lyophilization, the preformulationwas diluted 1-fold, ½-fold and ¼-fold using distilled water. Thelyophilization consisted of primary drying and secondary drying steps.The temperature gradient of the lyophilization is shown in FIG. 1. Thereconstitution was conducted by dissolving the lyophilized formulationwith distilled water of the same volume as that of the formulationbefore the lyophilization. The reconstitution was performed using anauto shaker set to 60° and 30 rpm. The time required for completedissolution is given in Table 11.

TABLE 10 Sugar alcohol and other Conc. Buffer Salt stabilizer SurfactantEx. 20 39.0 20 mM sodium — 5% mannitol 0.005% mg/mL citrate (pH 5.2) 5mM histidine polysorbate 80 Ex. 21 48.8 20 mM sodium — 5% mannitol0.005% mg/mL citrate (pH 5.2) 5 mM histidine polysorbate 80 Ex. 22 58.520 mM sodium — 5% mannitol 0.005% mg/mL citrate (pH 5.2) 5 mM histidinepolysorbate 80

TABLE 11 Example 20 Example 21 Example 22 Dilution factor 1 ½ ¼ 1 ½ ¼ 1½ ¼ Dissolution time (sec) 10 10 5 15 10 5 15 10 10

As seen from Table 11, it was confirmed that the dissolution rate isimproved when the density of the lyophilized substance is decreasedthrough dilution. Also, the dissolution rate increased similarly whenthe concentration of the long-acting hGH conjugate in the formulation(20 mM sodium citrate, 5% mannitol, 5 mM histidine, 0.005% polysorbate80) was increased from 39.0 mg/mL to 48.8 mg/mL and to 58.5 mg/mL.

(6) Setting of Temperature Gradient for Lyophilization Process

The temperature gradient in Test Example 1-(1) (FIG. 1) was changed byincreasing the primary drying time from 10 hours to 20 hours andsubdividing the temperature of the primary drying step (4° C.) into twostages of −20° C. and −5° C. (FIG. 2). In the former temperaturegradient, disruption of the lyophilized substance occurred because 3-5%of water remained in the lyophilized substance. When the temperaturegradient was changed to that shown in FIG. 2, complete lyophilizationcould be achieved even with a larger volume (˜5 mL).

(7) Analysis of Solubility of Lyophilized Formulation of Long-Acting hGHConjugate Considering Osmotic Pressure

Using the formulation (20 mM sodium acetate, pH 5.6, 5% (w/v) mannitol,150 mM sodium chloride, 0.005% (w/v) polysorbate 80) of Test Example1-(1) and (2), the concentration of the stabilizer was set consideringosmotic pressure and the solubility of the lyophilized substance wasanalyzed. Preformulations were prepared as described in Table 12 andthen lyophilized.

During the lyophilization, the preformulation was diluted ½-fold usingdistilled water. The lyophilization consisted of primary drying andsecondary drying steps. The temperature gradient of the lyophilizationwas set as shown in FIG. 2. The reconstitution was conducted usingdistilled water containing 0.9% benzyl alcohol, which has the samevolume as that of the formulation before the dilution.

The reconstitution was performed using an auto shaker set to 60° and 30rpm. The time required for complete dissolution and the osmotic pressuremeasured after the reconstitution are shown in Table 13.

TABLE 12 Sugar alcohol and other Conc. Buffer Salt stabilizer SurfactantEx. 23 58.5 mg/mL 20 mM sodium 150 mM   5% 0.005% acetate (pH NaClmannitol polysorbate 5.6) 80 Ex. 24 58.5 mg/mL 20 mM sodium 150 mM —0.005% acetate (pH NaCl polysorbate 5.6) 80 Ex. 25 58.5 mg/mL 20 mMsodium —   5% 0.005% acetate (pH mannitol polysorbate 5.6) 80 Ex. 2658.5 mg/mL 20 mM sodium  75 mM 2.5% 0.005% acetate (pH NaCl mannitolpolysorbate 5.6) 80

TABLE 13 Example 23 Example 24 Example 25 Example 26 Dissolution time 30180 15 60 (sec) Osmotic pressure 662 338 365 355 (mOsm/Kg)

As seen from Table 13, the dissolution time was increased greatly whenmannitol was removed. The osmotic pressure was higher than the isotonicrange of 280-320 mOsm/Kg when the concentration of mannitol was 5%(w/v).

(8) Analysis of Solubility and Osmotic Pressure of LyophilizedFormulation of Long-Acting hGH Conjugate Depending on MannitolConcentration

Using the formulation (20 mM sodium acetate, pH 5.6, 5% (w/v) mannitol,0.005% (w/v) polysorbate 80) of Test Example 1-(7), the solubility andosmotic pressure of the lyophilized substance depending on mannitolconcentration were analyzed.

Preformulations were prepared as described in Table 14 and thenlyophilized. The methods and conditions of the lyophilization andreconstitution were the same as described in Test Example 1-(7). Thetime required for complete dissolution and the osmotic pressure measuredafter the reconstitution are shown in Table 15.

TABLE 14 Sugar alcohol and other Conc. Buffer Salt stabilizer SurfactantEx. 23 58.5 20 mM sodium 150 mM   5% 0.005% mg/mL acetate (pH NaClmannitol polysorbate 5.6) 80 Ex. 25 58.5 20 mM sodium —   5% 0.005%mg/mL acetate (pH mannitol polysorbate 5.6) 80 Ex. 27 58.5 20 mM sodium— 4.5% 0.005% mg/mL acetate (pH mannitol polysorbate 5.6) 80 Ex. 28 58.520 mM sodium —   4% 0.005% mg/mL acetate (pH mannitol polysorbate 5.6)80 Ex.29 58.5 20 mM sodium — 3.5% 0.005% mg/mL acetate (pH mannitolpolysorbate 5.6) 80

TABLE 15 Ex. 23 Ex. 25 Ex. 27 Ex. 28 Ex. 29 Dissolution time 30 20 22 2527 (sec) osmotic pressure 632 350 318 291 258 (mOsm/Kg)

As seen from Table 15, isotonic osmotic pressure was observed when theconcentration of mannitol was in the range from 4 to 4.5%. Although thedissolution time was longer as the mannitol concentration was lower, thechange was smaller as compared to when sodium chloride was included.

(9) Analysis of Storage Stability of Lyophilized Formulation ofLong-Acting hGH Conjugate at 4° C. and 25° C.

Using the formulation (20 mM sodium acetate, pH 5.6, 4% (w/v) mannitol,0.005% (w/v) polysorbate 80) of Test Example 1-(8), the storagestability of the lyophilized substance was analyzed at 4° C. and 25° C.After storing the lyophilized formulation at 4° C. and 25° C. for 6months, the stability was evaluated by ion exchange chromatography(IE-HPLC) after reconstitution. The initial solution for reconstitutionwas stored at 25° C. for 4 weeks in liquid state and then was evaluatedby ion exchange chromatography (IE-HPLC) after reconstitution. In Table16, IE-HPLC (%) indicates the purity of the long-acting hGH conjugate inthe lyophilized substance at the given time. In Table 17, IE-HPLC (%)indicates the purity of the long-acting hGH conjugate in thereconstituted liquid formulation.

TABLE 16 IE-HPLC (%) Month 0 Month 3 Month 6  4° C. 96.6 96.5 96.2 25°C. 96.5 95.6 96.0

TABLE 17 IE-HPLC (%) Week 0 Week 1 Week 2 Week 4 96.6 93.1 89.4 87.7

As seen from Table 16, the stability of the lyophilized substance wasmaintained even after storing at 4° C. and 25° C. for 6 months. Also,the stability of the reconstituted formulation was maintained even afterstoring at 25° C. for 2 weeks.

Test Example 2 Evaluation of Liquid Formulation of Long-Acting hGHConjugate

(1) Analysis of Stability of Liquid Formulation of Long-Acting hGHConjugate Depending on pH, Buffer, Isotonic Agent and Sugar AlcoholConcentration

The effect of a buffer, an isotonic agent and the sugar alcoholconcentration on the stability of the long-acting hGH conjugate wastested. Formulations prepared as described in Table 18 were stored at25° C. for 0-4 weeks and then analyzed by ion exchange chromatographyand size exclusion chromatography. In Tables 19 and 20, IE-HPLC (%) andSE-HPLC (%) indicate the residual rate of the long-acting hGH conjugaterelative to the initial value, respectively (area %/start area %).

TABLE 18 Sugar Long-acting Iso- alcohol hGH tonic and other conjugate pHBuffer agent stabilizer Surfactant Ex. 30 58.5 mg/mL 5.2 20 mM 75 mM 2%0.005% sodium NaCl mannitol polysorbate acetate 80 Ex. 31 58.5 mg/mL 5.220 mM — 4% 0.005% sodium mannitol polysorbate acetate 80 Ex. 32 58.5mg/mL 5.6 20 mM 75 mM 2% 0.005% sodium NaCl mannitol polysorbate acetate80 Ex. 33 58.5 mg/mL 5.6 20 mM — 4% 0.005% sodium mannitol polysorbateacetate 80 Ex. 34 58.5 mg/mL 5.6 20 mM 75 mM 2% 0.005% histi- NaClmannitol polysorbate dine 80 Ex. 35 58.5 mg/mL 5.6 20 mM — 4% 0.005%histi- mannitol polysorbate dine 80

TABLE 19 IE-HPLC (%) Week 0 Week 1 Week 2 Week 3 Week 4 Example 30 100.098.5 97.0 95.3 91.9 Example 31 100.0 98.9 96.9 94.8 91.9 Example 32100.0 96.9 94.9 92.2 87.2 Example 33 100.0 97.8 96.7 94.7 90.2 Example34 100.0 97.8 94.7 92.4 86.5 Example 35 100.0 96.1 93.0 88.4 82.9

TABLE 20 SE-HPLC (%) Week 0 Week 1 Week 2 Week 3 Week 4 Example 30 100.099.6 99.4 99.3 99.1 Example 31 100.0 100.2 100.0 99.9 99.8 Example 32100.0 99.6 99.6 99.5 99.4 Example 33 100.0 99.6 99.6 99.4 99.3 Example34 100.0 100.1 100.1 99.9 99.7 Example 35 100.0 99.9 99.8 99.7 99.3

The IE-HPLC result showed that the long-acting hGH conjugate shows goodstability under the condition of 20 mM sodium acetate (pH 5.2). And, theSE-HPLC result showed that the long-acting hGH conjugate was the moststable under the condition of 20 mM sodium acetate (pH 5.2) and 4% (w/v)mannitol.

(2) Analysis of Stability of Liquid Formulation of a Long-Acting hGHConjugate Depending on Buffer

The effect of a buffer as a stabilizer on the stability of thelong-acting hGH conjugate was tested. Using the formulation (pH 5.2, 4%mannitol, 0.005% polysorbate 80) of Test Example 2-(1), formulationswere prepared as described in Table 21. After storing at 25° C. for 0-4weeks, the stability was analyzed by IE-HPLC and SE-HPLC. In Tables 22and 23, IE-HPLC (%) and SE-HPLC (%) indicate the residual rate of thelong-acting hGH conjugate relative to the initial value, respectively(area %/start area %).

TABLE 21 Sugar Long-acting Iso- alcohol hGH tonic and other conjugate pHBuffer agent stabilizer Surfactant Ex. 36 10.0 mg/mL 5.2 20 mM — 4%mannitol 0.005% sodium polysorbate citrate 80 Ex. 37 10.0 mg/mL 5.2 20mM — 4% mannitol 0.005% sodium polysorbate acetate 80 Ex. 38 10.0 mg/mL5.2 20 mM — 4% mannitol 0.005% histi- polysorbate dine 80 Ex. 39 58.5mg/mL 5.2 20 mM — 4% mannitol 0.005% sodium polysorbate citrate 80 Ex.31 58.5 mg/mL 5.2 20 mM — 4% mannitol 0.005% sodium polysorbate acetate80 Ex. 40 58.5 mg/mL 5.2 20 mM — 4% mannitol 0.005% histi- polysorbatedine 80

TABLE 22 IE-HPLC (%) Week 0 Week 1 Week 2 Week 3 Week 4 Example 36 100.097.4 96.1 94.9 93.1 Example 37 100.0 98.3 96.5 92.7 88.5 Example 38100.0 97.9 96.4 93.9 91.7 Example 39 100.0 98.4 96.7 94.8 92.7 Example31 100.0 97.9 96.3 94.6 92.8 Example 40 100.0 98.2 96.3 94.2 92.2

TABLE 23 SE-HPLC (%) Week 0 Week 1 Week 2 Week 3 Week 4 Example 36 100.098.9 98.7 98.7 98.4 Example 37 100.0 98.6 98.6 98.8 98.1 Example 38100.0 98.7 98.8 98.8 98.6 Example 39 100.0 99.4 99.2 99.2 98.9 Example31 100.0 99.8 99.1 99.2 98.8 Example 40 100.0 100.0 99.2 99.4 99.1

The IE-HPLC result showed that 10.0 mg/mL long-acting hGH conjugate wasthe most stable under the condition of sodium citrate. And, the SE-HPLCresult showed that 10.0 mg/mL long-acting hGH conjugate showed goodstability in the order of histidine and sodium citrate.

The IE-HPLC result showed that 58.5 mg/mL long-acting hGH conjugateshowed good stability in the order of sodium acetate and sodium citrate.And, the SE-HPLC result showed that 58.5 mg/mL long-acting hGH conjugateshowed good stability in the order of histidine and sodium citrate.

When the concentration of the long-acting hGH conjugate was in the rangefrom 10 mg/mL to 58.5 mg/mL, the best stability was observed under thecondition of 20 mM sodium citrate (pH 5.2), 4% mannitol and 0.005%polysorbate 80.

It will be apparent to those skilled in the art that variousmodifications and changes may be made without departing from the scopeand spirit of the invention. Therefore, it should be understood that theabove embodiment is not limitative, but illustrative in all aspects. Thescope of the invention is defined by the appended claims rather than bythe description preceding them, and therefore all changes andmodifications that fall within metes and bounds of the claims, orequivalents of such metes and bounds are therefore intended to beembraced by the claims.

The invention claimed is:
 1. A liquid formulation of a long-acting humangrowth hormone (hGH) conjugate, wherein the liquid formulation comprisesa pharmaceutically effective amount of a long-acting human growthhormone conjugate in which the physiologically active human growthhormone is linked to an immunoglobulin Fc region and an albumin-freestabilizer, wherein the liquid formulation comprises a buffer, anon-ionic surfactant, a sugar alcohol, and preservative, wherein thesugar alcohol is included with a concentration ranging from 2% (w/v) to4.5% (w/v); wherein the preservative is benzyl alcohol, m-cresol, orphenol; wherein the formulation does not comprise sodium chloride;wherein the buffer is a citrate buffer, an acetate buffer, or ahistidine buffer; wherein the non-ionic surfactant is polysorbate 80;wherein the sugar alcohol is mannitol or sorbitol; and wherein theconcentration of the long-acting hGH conjugate ranges from 58.5 to 60mg/mL.
 2. The liquid formulation of a long-acting human growth hormoneconjugate according to claim 1, wherein the pH of the buffer ranges from5.0 to 6.0.
 3. The liquid formulation of a long-acting human growthhormone conjugate according to claim 1, wherein the concentration of thenon-ionic surfactant ranges from 0.001% (w/v) to 0.05% (w/v) of thetotal volume of the formulation.
 4. The liquid formulation of along-acting human growth hormone conjugate according to claim 1, whereinthe conjugate is in such a form that the human growth hormone is linkedto the immunoglobulin Fc via a non-peptidyl polymer as a linker or viagenetic recombination.
 5. The liquid formulation of a long-acting humangrowth hormone conjugate according to claim 4, wherein the non-peptidylpolymer is a biodegradable polymer.
 6. The liquid formulation of along-acting human growth hormone conjugate according to claim 5, whereinthe biodegradable polymer is selected from the group consisting ofpolyethylene glycol, polypropylene glycol, a copolymer of ethyleneglycol and propylene glycol, a polyoxyethylated polyol, polyvinylalcohol, a polysaccharide, dextran, polyvinyl ethyl ether, polylacticacid (PLA) and polylactic-glycolic acid (PLGA), a lipid polymer, chitin,hyaluronic acid and a combination thereof.